Compositions and methods for treating bacteria

ABSTRACT

The present invention relates to the field of bacteriology. In particular, the present invention provides compositions (e.g., comprising a lantibiotic and mupirocin or gentamicin) and methods of treating (e.g., killing or inhibiting growth of) bacteria. For example, the present invention provides pharmaceutical compounds (e.g., comprising a lantibiotic and mupirocin or gentamicin) and methods of using the same in research, therapeutic and drug screening applications.

This application is a Divisional application of U.S. patent applicationSer. No. 11/494,887 filed Jul. 28, 2006, which claims priority to U.S.Provisional Patent App. Nos. 60/703,010 filed Jul. 28, 2005; 60/725,193,filed Oct. 11, 2005; and 60/779,034, filed Mar. 3, 2006, each of whichis hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of bacteriology. Inparticular, the present invention provides compositions (e.g.,comprising a lantibiotic and mupirocin and/or gentamicin) and methods oftreating (e.g., killing or inhibiting growth of) bacteria. For example,the present invention provides pharmaceutical compounds (e.g.,comprising a lantibiotic and mupirocin and/or gentamicin) and methods ofusing the same in research, therapeutic and drug screening applications.

BACKGROUND OF THE INVENTION

As the use of conventional pharmaceutical antibiotics has increased formedical, veterinary and agricultural purposes, there has been aconcurrent emergence of antibiotic-resistant strains of pathogenicbacteria.

The emergence of single- or multi-drug resistant bacteria can resultfrom a gene mobilization that responds to selective pressures associatedwith antibiotic use. Over the last several decades, the increasinglyfrequent usage of antibiotics has acted in concert with spontaneousmutations arising in the bacterial gene pool to produce differentstrains of bacteria not susceptible to current antibacterial treatments.This repertoire of antibiotic resistant genes can be utilized bypreviously sensitive strains that have access to these genes (e.g., viaconjugative transfer of plasmids or transposons). As a result, single-and multi-drug resistance genes are commonly found in a large variety ofbacterial plasmids and conjugative transposons.

Gram-positive bacteria are a major cause of nosocomial infection. Themost common pathogenic isolates in hospitals include Enterococcus spp.,Staphylococcus aureus, coagulase-negative staphylococci, andStreptococcus pneumoniae (See, e.g., Principles and Practice ofInfectious Diseases, 4th ed. Mandell G L, Bennett J E, Dolin R, ed.Churchill Livingstone, New York 1995), many strains of which areresistant to one or more antibiotics. Enterococcus spp. are part of thenormal gut flora in humans. Of the more than seventeen enterococcalspecies, only E. faecalis and E. faecium commonly colonize and infecthumans in detectable numbers (E. faecalis is isolated from approximately80% of human infections, and E. faecium from most of the rest).

Vancomycin-resistant enterococcus (VRE) spp. are becoming increasinglycommon in hospital settings. In the first half of 1999, 25.9% ofentercoccal isolates from Intensive Care Units werevancomycin-resistant; an increase from 16.6% in 1996 and from 0.4% in1989. VRE are also commonly resistant to many other commercialantibiotics, including beta-lactams and aminoglycosides. Thus, patientswho are immunocompromised or those having a prolonged hospital stay areat increased risk for acquiring a VRE infection.

The problem of antibiotic resistance is not unique to Enterococcus spp.Strains of many other potentially pathogenic Gram-positive bacteriadisplaying antibiotic resistance have been isolated includingmethicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistantStaphylococcus aureus (VRSA), glycopeptide intermediate-susceptibleStaphylococcus aureus (GISA), vancomycin-resistant MRSA (VR-MRSA) andpenicillin-resistant Streptococcus pneumoniae (PRSP). Like VRE,therapeutic options for treating infections by these organisms arelimited.

Resistance transfer is another complicating factor in the management ofantibiotic-resistant infections. Vancomycin resistance can transfer fromVRE to other Gram-positive bacteria, including S. aureus, in vitro.Thus, the presence of resistant bacteria (e.g., VRE) in a hospital posesnot just the risk of infection but also the continued evolution ofresistant organisms (e.g., creating more virulent organisms such asVR-MRSA).

A need exists to develop alternative strategies of antibacterialtreatment. For example, there exists a need for new compositions andmethods of treating or preventing bacterial infection (e.g., bacteremia)caused by strains of bacteria unsusceptible to current forms ofantibacterial treatments (e.g., Gram-positive bacteria such as MRSA andVRE).

SUMMARY OF THE INVENTION

The present invention relates to the field of bacteriology. Inparticular, the present invention provides compositions (e.g.,comprising a lantibiotic and mupirocin and/or gentamicin) and methods oftreating (e.g., killing or inhibiting growth of) bacteria. For example,the present invention provides pharmaceutical compounds (e.g.,comprising a lantibiotic and mupirocin and/or gentamicin) and methods ofusing the same in research, therapeutic and drug screening applications.

Accordingly, in some embodiments, the present invention providescompositions and methods for the treatment of bacteria (e.g., bacterialinfection). In some preferred embodiments, the present inventionprovides a combinatorial treatment for bacteria (e.g., a treatment thatinhibits growth of and/or that kills bacteria). For example, in someembodiments, the present invention provides a composition comprising ananti-infective peptide (i.e., a peptide that inhibits growth or that iscapable of killing bacteria (e.g., Gram positive bacteria)) and othertype of anti-infective pharmaceutical or compound (e.g., non peptideanti-infective). In some embodiments, the anti-infective peptide is acationic peptide. In some embodiments, the anti-infective peptide is adefensin. In some embodiments, the anti-infective peptide is alantibiotic (e.g., nisin). In some embodiments, the secondanti-infective agent is a small molecule antibiotic. In someembodiments, the second anti-infective agent is an antibiotic thatalters bacterial metabolism (e.g., an antibiotic that interferes withcellular metabolism and/or RNA synthesis and/or protein synthesis). Insome embodiments, the second anti-infective agent is an amino glycoside(e.g., gentimycin). In some embodiments, the second anti-infective agentis mupirocin. Although an understanding of the mechanism is notnecessary to practice the present invention and the present invention isnot limited to any particular mechanism of action, it is contemplatedthat a composition comprising an anti-infective peptide is capable ofgenerating pores in bacteria thereby enhancing the entry of compounds(e.g., small molecule antibiotic or metabolism antibiotic) intobacteria. Although an understanding of the mechanism is not necessary topractice the present invention and the present invention is not limitedto any particular mechanism of action, it is contemplated that thecompositions of the present invention may stimulate (e.g., when incontact with the skin) a host defense response (e.g., an innate immuneresponse) thereby decreasing the likelihood of or reducing bacterialinfection.

In some embodiments, the present invention provides a topicalpharmaceutical formulation comprising a lantibiotic and mupirocin. Insome embodiments, the lantibiotic is nisin. However, the presentinvention is not limited by the type of lantibiotic utilized. Indeed, anumber of lantibiotics may be used including, but not limited to,subtilin, epidermin, gallidermin, pep 5, cinnamycin, duramycin andancovenin. In some embodiments, the pharmaceutical formulation is acream. In some embodiments, the pharmaceutical formulation is a spray.In some embodiments, the pharmaceutical formulation is a gel or othertype of formulation useful for administration of the lantibiotic andmupirocin. In some embodiments, the pharmaceutical formulation comprisespolyethylene glycol. In some embodiments, the pharmaceutical formulationis prepared for controlled time release.

The present invention also provides a method of altering growth ofbacteria comprising administrating to the bacteria a compositioncomprising a lantibiotic and mupirocin. In some embodiments, alteringgrowth of bacteria comprises killing the bacteria. In some embodiments,altering growth of bacteria comprises inhibiting growth of the bacteria.In some embodiments, altering growth of bacteria comprises a 3 log orgreater reduction of bacterial cell presence. In some embodiments, thelantibiotic is nisin. In some embodiments, the bacteria are pathogenicbacteria. In some embodiments, the bacteria are from the genusStaphylococci. In some embodiments, the bacteria are Staphylococcusaureus. In some embodiments, the bacteria are Staphylococcusepidermidis. In some embodiments, the Staphylococci are antibioticresistant. For example, in some embodiments, the Staphylococcus aureusare methicillin resistant or vancomycin resistant. In some embodiments,the composition is a topical pharmaceutical formulation.

The present invention also provides a method for treating or preventinginfection comprising exposing a surface of a subject to a topicalpharmaceutical formulation comprising a lantibiotic and mupirocin. Insome embodiments, exposing a surface of a subject to a topicalpharmaceutical formulation comprising a lantibiotic and mupirocin treatsor prevents growth of Staphylococcus aureus on the surface of thesubject. The present invention is not limited by the type of surfacetreated. Indeed, a variety of surfaces may be treated including, but notlimited to, skin surfaces (e.g., epidermis or sub-epidermal layers),wounds and cuts (e.g., superficial wounds, partial thickness wounds anddeep cuts), mucosal surfaces, etc). The present invention is not limitedby the type of subject treated. Indeed, a variety of subjects may betreated including, but not limited to, humans, large and small mammals,rodents, fish, etc. In some embodiments, the topical formulationcomprises a cream. In some embodiments, treating results in a 3 log orgreater reduction in bacteria responsible for the infection. In someembodiments, treating results in a lack of detectable bacteriaresponsible for the infection. In some embodiments, the reduction occurswithin three days of treating. In some embodiments, the reduction occurswith two days of treating. In some embodiments, the lantibiotic isnisin. In some embodiments, the lantibiotic is gallidermin.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the efficacy of nisin alone, mupirocin alone, and thecombination of nisin and mupirocin on S. aureus infection in abradedmouse skin.

FIG. 2 shows the efficacy of nisin, lysostaphin, and bacitracin aloneand in combination on S. aureus infection in abraded mouse skin usingtwo different formulations of bacitracin.

FIG. 3 shows the efficacy of nisin alone, mupirocin alone, and thecombination of nisin and mupirocin on mupirocin-resistant S. aureusinfection in abraded mouse skin.

FIG. 4 shows the efficacy of nisin and mupirocin in combination withEDTA on P. aeruginosa infection in abraded mouse skin.

FIG. 5 depicts the experimental design of using compositions and methodsof the present invention to treat partial thickness wounds.

FIG. 6 shows Staphylococcus aureus growth in inoculated wounds two daysafter treatment with various agents.

FIG. 7 shows Staphylococcus aureus growth in inoculated wounds five daysafter treatment with various agents.

FIG. 8 shows Staphylococcus aureus growth in inoculated wounds sevendays after treatment with various agents.

FIG. 9 shows a composite of Staphylococcus aureus growth in inoculatedwounds two, five and seven days after treatment with various agents.

FIG. 10 shows the mean log (CFU/ml) of bacteria recovered after either afirst or second treatment (MSSA) or after one and three days (MRSA) oftreatment with the reagents indicated.

FIG. 11 shows the minimum inhibitory concentration (MIC) of treatment ofS. aureus in vitro with nisin alone or nisin in combination with eithermupirocin, neomycin or gentamicin.

FIG. 12 shows that mupirocin does not synergize with or provide additivebenefit together with bacitracin in treating skin infection.

FIG. 13 shows that nisin and mupirocin function synergistically to treatS. aureus in a suture infection mouse model.

FIG. 14 shows data representing the log colony forming units/mLdetermined from cultured wounds 2, 5 and 7 days post treatment withcompositions of the present invention.

DEFINITIONS

As used herein, the term “subject” refers to an individual (e.g., human,animal, or other organism) to be treated by the methods or compositionsof the present invention. Subjects include, but are not limited to,mammals (e.g., murines, simians, equines, bovines, porcines, canines,felines, and the like), and most preferably includes humans. In thecontext of the invention, the term “subject” generally refers to anindividual who will receive or who has received treatment for acondition characterized by the presence of bacteria (e.g., pathogenicbacteria such as MRSA), or in anticipation of possible exposure tobacteria. As used herein, the terms “subject” and “patient” are usedinterchangeably, unless otherwise noted.

The term “diagnosed,” as used herein, refers to the recognition of adisease (e.g., caused by the presence of pathogenic bacteria) by itssigns and symptoms (e.g., resistance to conventional therapies), orgenetic analysis, pathological analysis, histological analysis, and thelike.

As used herein the term, “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments include, but are not limited to, testtubes and cell cultures. The term “in vivo” refers to the naturalenvironment (e.g., an animal or a cell) and to processes or reactionthat occur within a natural environment.

As used herein, the terms “attenuate” and “attenuation” used inreference to a feature (e.g. growth) of a bacterial cell or a populationof bacterial cells refers to a reduction, inhibition or elimination ofthat feature, or a reducing of the effect(s) of that feature.

As used herein, the term “effective amount” refers to the amount of acomposition (e.g., a composition comprising mupirocin and nisin)sufficient to effect a beneficial or desired result (e.g., bacterialcell killing). An effective amount can be administered in one or moreadministrations, applications or dosages and is not intended to belimited to a particular formulation or administration route.

As used herein, the term “administration” refers to the act of giving adrug, prodrug, or other agent, or therapeutic treatment (e.g., acomposition of the present invention) to a physiological system (e.g., asubject or in vivo, in vitro, or ex vivo cells, tissues, and organs).Exemplary routes of administration to the human body can be through theeyes (ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs(inhalant), mucosal (e.g.,oral mucosa or buccal), rectal, ear, byinjection (e.g., intravenously, subcutaneously, intratumorally,intraperitoneally, etc.) and the like.

As used herein, the term “treating a surface” refers to the act ofexposing a surface to one or more compositions of the present invention.Methods of treating a surface include, but are not limited to, spraying,misting, submerging, and coating. Surfaces include organic surfaces(e.g., food products, surfaces of animals, etc.) and inorganic surfaces(e.g., medical devices, countertops, clothing, etc.).

As used herein, the term “co-administration” refers to theadministration of at least two agent(s) or therapies to a subject. Insome embodiments, the co-administration of two or more agents ortherapies is concurrent. In other embodiments, a first agent/therapy isadministered prior to a second agent/therapy. Those of skill in the artunderstand that the formulations and/or routes of administration of thevarious agents or therapies used may vary. The appropriate dosage forco-administration can be readily determined by one skilled in the art.In some embodiments, when agents or therapies are co-administered, therespective agents or therapies are administered at lower dosages thanappropriate for their administration alone. Thus, co-administration isespecially desirable in embodiments where the co-administration of theagents or therapies lowers the requisite dosage of a potentially harmful(e.g., toxic) agent(s).

As used herein, the term “toxic” refers to any detrimental or harmfuleffects on a subject, a cell, or a tissue as compared to the same cellor tissue prior to the administration of the toxicant.

As used herein, the term “pharmaceutical composition” refers to thecombination of an active agent (e.g., a composition comprising mupirocinand nisin) with a carrier, inert or active, making the compositionespecially suitable for diagnostic or therapeutic use in vitro, in vivoor ex vivo.

The terms “pharmaceutically acceptable” or “pharmacologicallyacceptable,” as used herein, refer to compositions that do notsubstantially produce adverse reactions (e.g., toxic, allergic, orimmunological reactions) when administered to a subject.

As used herein, the term “topically” refers to application of thecompositions of the present invention to the surface of the skin ormucosal cells and tissues (e.g., alveolar, buccal, lingual, masticatory,or nasal mucosa, and other tissues and cells that line hollow organs orbody cavities).

As used herein, the term “pharmaceutically acceptable carrier” refers toany of the standard pharmaceutical carriers including, but not limitedto, phosphate buffered saline solution, water, emulsions (e.g., such asan oil/water or water/oil emulsions), and various types of wettingagents, any and all solvents, dispersion media, coatings, sodium laurylsulfate, isotonic and absorption delaying agents, disintrigrants (e.g.,potato starch or sodium starch glycolate), and the like. Thecompositions also may include stabilizers and preservatives. Examples ofcarriers, stabilizers, and adjuvants are described in the art (See e.g.,Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co.,Easton, Pa. (1975), incorporated herein by reference).

As used herein, the term “medical devices” includes any material ordevice that is used on, in, or through a subject's or patient's body,for example, in the course of medical treatment (e.g., for a disease orinjury). Medical devices include, but are not limited to, such items asmedical implants, wound care devices, drug delivery devices, and bodycavity and personal protection devices. The medical implants include,but are not limited to, urinary catheters, intravascular catheters,dialysis shunts, wound drain tubes, skin sutures, vascular grafts,implantable meshes, intraocular devices, heart valves, and the like.Wound care devices include, but are not limited to, general wounddressings, biologic graft materials, tape closures and dressings, andsurgical incise drapes. Drug delivery devices include, but are notlimited to, needles, drug delivery skin patches, drug delivery mucosalpatches and medical sponges. Body cavity and personal protectiondevices, include, but are not limited to, tampons, sponges, surgical andexamination gloves, and toothbrushes. Birth control devices include, butare not limited to, intrauterine devices (IUDs), diaphragms, andcondoms.

As used herein, the term “therapeutic agent” refers to a compositionthat decreases the infectivity, morbidity, or onset of mortality in asubject contacted by a pathogenic microorganism or that preventinfectivity, morbidity, or onset of mortality in a host contacted by apathogenic microorganism. Therapeutic agents encompass agents usedprophylactically (e.g., in the absence of a pathogen) in view ofpossible future exposure to a pathogen. Such agents may additionallycomprise pharmaceutically acceptable compounds (e.g., adjuvants,excipients, stabilizers, diluents, and the like). In some embodiments,the therapeutic agents of the present invention are administered in theform of topical compositions, injectable compositions, ingestiblecompositions, and the like. When the route is topical, the form may be,for example, a solution, cream, ointment, salve or spray.

As used herein, the term “pathogen” refers a biological agent thatcauses a disease state (e.g., infection, sepsis, etc.) in a host.“Pathogens” include, but are not limited to, viruses, bacteria, archaea,fungi, protozoans, mycoplasma, prions, and parasitic organisms.

The terms “bacteria” and “bacterium” refer to all prokaryotic organisms,including those within all of the phyla in the Kingdom Procaryotae. Itis intended that the term encompass all microorganisms considered to bebacteria including Mycoplasma, Chlamydia, Actinomyces, Streptomyces, andRickettsia. All forms of bacteria are included within this definitionincluding cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc.Also included within this term are prokaryotic organisms that areGram-negative or Gram-positive. “Gram-negative” and “Gram-positive”refer to staining patterns with the Gram-staining process, which is wellknown in the art. (See e.g., Finegold and Martin, DiagnosticMicrobiology, 6th Ed., CV Mosby St. Louis, pp. 13-15 (1982)).“Gram-positive bacteria” are bacteria that retain the primary dye usedin the Gram stain, causing the stained cells to generally appear darkblue to purple under the microscope. “Gram-negative bacteria” do notretain the primary dye used in the Gram stain, but are stained by thecounterstain. Thus, Gram-negative bacteria generally appear red. In someembodiments, bacteria are continuously cultured. In some embodiments,bacteria are uncultured and existing in their natural environment (e.g.,at the site of a wound or infection) or obtained from patient tissues(e.g., via a biopsy). Bacteria may exhibit pathological growth orproliferation. Examples of bacteria include, but are not limited to,bacterial cells of a genus of bacteria selected from the groupcomprising Salmonella, Shigella, Escherichia, Enterobacter, Serratia,Proteus, Yersinia, Citrobacter, Edwardsiella, Providencia, Klebsiella,Hafnia, Ewingella, Kluyvera, Morganella, Planococcus, Stomatococcus,Micrococcus, Staphylococcus, Vibrio, Aeromonas, Plessiomonas,Haemophilus, Actinobacillus, Pasteurella, Mycoplasma, Ureaplasma,Rickettsia, Coxiella, Rochalimaea, Ehrlichia, Streptococcus,Enterococcus, Aerococcus, Gemella, Lactococcus, Leuconostoc, Pedicoccus,Bacillus, Corynebacterium, Arcanobacterium, Actinomyces, Rhodococcus,Listeria, Erysipelothrix, Gardnerella, Neisseria, Campylobacter,Arcobacter, Wolinella, Helicobacter, Achromobacter, Acinetobacter,Agrobacterium, Alcaligenes, Chryseomonas, Comamonas, Eikenella,Flavimonas, Flavobacterium, Moraxella, Oligella, Pseudomonas,Shewanella, Weeksella, Xanthomonas, Bordetella, Franciesella, Brucella,Legionella, Afipia, Bartonella, Calymmatobacterium, Cardiobacterium,Streptobacillus, Spirillum, Peptostreptococcus, Peptococcus, Sarcinia,Coprococcus, Ruminococcus, Propionibacterium, Mobiluncus,Bifidobacterium, Eubacterium, Lactobacillus, Rothia, Clostridium,Bacteroides, Porphyromonas, Prevotella, Fusobacterium, Bilophila,Leptotrichia, Wolinella, Acidaminococcus, Megasphaera, Veilonella,Norcardia, Actinomadura, Norcardiopsis, Streptomyces, Micropolysporas,Thermoactinomycetes, Mycobacterium, Treponema, Borrelia, Leptospira, andChlamydiae.

As used herein, the term “microorganism” refers to any species or typeof microorganism, including but not limited to, bacteria, archaea,fungi, protozoans, mycoplasma, and parasitic organisms.

As used herein, the term “non-human animals” refers to all non-humananimals including, but not limited to, vertebrates such as rodents,non-human primates, ovines, bovines, ruminants, lagomorphs, porcines,caprines, equines, canines, felines, aves, etc.

As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of reaction materials (e.g.,compositions comprising mupirocin and nisin), such delivery systemsinclude systems that allow for the storage, transport, or delivery ofreaction reagents and/or supporting materials (e.g., writteninstructions for using the materials, etc.) from one location toanother. For example, kits include one or more enclosures (e.g., boxes)containing the relevant reaction reagents and/or supporting materials.As used herein, the term “fragmented kit” refers to delivery systemscomprising two or more separate containers that each contain asubportion of the total kit components. The containers may be deliveredto the intended recipient together or separately. For example, a firstcontainer may contain a composition comprising mupirocin and nisin for aparticular use, while a second container contains a second agent (e.g.,an antibiotic or spray applicator). Indeed, any delivery systemcomprising two or more separate containers that each contains asubportion of the total kit components are included in the term“fragmented kit.” In contrast, a “combined kit” refers to a deliverysystem containing all of the components of a reaction materials neededfor a particular use in a single container (e.g., in a single boxhousing each of the desired components). The term “kit” includes bothfragmented and combined kits.

DETAILED DESCRIPTION OF THE INVENTION

Staphylococci are gram positive bacterial pathogens that cause a widevariety of diseases ranging from superficial abscesses (e.g., boils,styes, furuncles and other localized abscesses) to deeper infections(e.g., osteomyelitis, pneumonia, endocarditis, urinary tract infections,septic arthritis, meningitis, post-operative wound infections,septicemia and food poisoning). S. aureus is a major cause of hospitalacquired (nosocomial) infection of surgical wounds and S. epidermidiscauses infections associated with indwelling medical devices. (See,e.g., Silverstein et al., 1990; Patti et al., 1994; Dann et al., 1994.)Multiple antibiotic resistance is increasingly common in S. aureus andS. epidermidis. Hospital strains of Staphylococcus are often resistantto many different antibiotics. S epidermidis nosocomial isolates arealso often resistant to several antibiotics including methicillin. Inaddition, S. aureus expresses resistance to antiseptics anddisinfectants, such as quaternary ammonium compounds, that may aid itssurvival in the hospital environment.

For serious hospital infections with multi-drug resistant S. aureus,vancomycin had existed as the only effective antibiotic. Vancomycinresistance is carried by conjugative plasmids that can be transferred toS. aureus in a laboratory setting (Noble et al., 1992) and has appearednaturally in enterococci (See, e.g., Arthur et al., 1993). However, theappearance of vancomycin resistant S. aureus has been reported (See,e.g., Lowry, 1998; Mathews et al., J Acquir Immune Defic Syndr. 2005 Oct1; 40(2):155-160) in the U.S. and abroad. Vaccines have been developedtargeting the organism or exotoxins it produces, but these approacheshave met with little success (See, e.g., Mamo et al., 1994),underscoring the need to develop new methods to control Staphylococcalinfections.

Similar to other gram positive bacteria, S. aureus causes diseasechiefly through the production of virulence factors such as hemolysins,enterotoxins and toxic shock syndrome toxin, which facilitate thesurvival, multiplication and spread of the organism in infected tissue(See, e.g., Mekalanos, 1992). The synthesis of most virulence factors inS. aureus is controlled by the accessory global regulon (agr) locus,which is activated by secreted autoinducing peptides (AIPs) (See, e.g.,Novick et al., 1993; Novick et al., 1995).

The presence of agr and regulation of virulence by RNAIII has beendemonstrated in all strains of S. aureus tested to date as well asseveral other species of staphylococci including S. epidermidis, S.lugdunesis, S. hemolyticus (See, e.g., Vandenesch et al., 1993), and S.warneri (See, e.g., Tegmark et al., 1998).

S. aureus infection remains one of the most common nosocomial andcommunity-acquired infections. With the continuing emergence ofmethicillin-resistant S. aureus (MRSA) and strains of S. aureus that areintermediately resistant to glycopeptides and the isolation of clinicalstrain of S. aureus that are fully vancomycin resistant, S. aureus isbecoming an even more difficult health problem to address, particularlyin settings such as hospitals and nursing homes.

Currently, BACTROBAN (2% mupirocin ointment; SmithKline Beecham,Bristol, Tenn.) is the most widely prescribed and effectiveantibacterial agent for treatment of S. aureus skin infections.Mupirocin is an antibacterial agent produced by fermentation using theorganism Pseudomonasfluorescens. It is active against a wide range ofgram-positive bacteria including most strains of S. aureus, includingmethicillin-resistant S. aureus (MRSA), most strains of S. epidermidis,S. saprophyticus, and Streptococcus. Mupirocin inhibits bacterialprotein synthesis by reversibly and specifically binding to bacterialisoleucyl transfer-RNA synthetase. Due to this unique mode of action,mupirocin demonstrates no in vitro cross-resistance with other classesof anti-microbial agents. Although BACTROBAN (2% mupirocin) is a commontreatment, a variety of compounds similar to mupirocin (e.g.,derivatives and functional equivalents) are in use or in development andare known in the art.

Unfortunately, as with many antimicrobials, mupirocin resistance isemerging in S. aureus and coagulase-negative Staphylococci. Furthermore,this antibiotic does not routinely eliminate all infectious organisms inall patients.

Nisin is an antimicrobial substance produced by Lactococcus lactisbelonging to the Lancefield serological group. It is a member of a groupof similar substances referred to as lantibiotics, that include, but arenot limited to, subtilin, epidermin, gallidermin, pep 5, cinnamycin,duramycin and ancovenin. Nisin is a peptide comprised of 34-amino acidresidues and contains five ring structures cross-linked by thioetherbridges that form lanthionine or methyllanthionine. Formulations ofnisin are described in U.S. Pat. Nos. 5,135,910 and 5,753,614, hereinincorporated by reference in their entireties. Variants of nisin aredescribed in U.S. Pat. No. 6,448,034, herein incorporated by referencein its entirety. Additional lantibiotics similar to nisin are describedin U.S. Pat. Nos. 5,594,103 and 5,928,146, herein incorporated byreference in their entireties.

Nisin has broad-spectrum activity against gram positive bacteria andsome activity against gram negative bacteria. Nisin has been used as anantimicrobial food preservative and is generally accepted as safe.Blackburn et al. (See U.S. Pat. No. 5,866,539, hereby incorporated byreference in its entirety) generally describes use of nisin along withanti-bacterial agents to treat skin infections.

Despite impressive successes in controlling or eliminating bacterialinfections by antibiotics, the widespread use of antibiotics both inhuman medicine and as a feed supplement in poultry and livestockproduction has led to drug resistance in many pathogenic bacteria. Theemergence of pan-resistant strains of Staphylococci (e.g., strains thatare unsusceptible to current forms of antibacterial treatment) makes theneed to control Staphylococcal infection an important medical concern.Thus, it is desirable to provide new compositions and methods oftreatment that display efficacy in reducing the incidence and severityof Staphylococcal infection. Specifically, there is a need for newtreatments for resistant strains of bacteria, in particular, S. aureusstrains that are resistant to mupirocin and other antibiotics. There isalso a need for anti-infectives that have a broader range of activityagainst gram negative bacteria, and for more effective anti-infectives.Additionally, it would be beneficial for such treatments to be welltolerated by patients with minimal or no side effects.

Accordingly, the present invention provides compositions and methods forthe treatment of bacteria (e.g., bacterial infection). In some preferredembodiments, the present invention provides a combinatorial treatmentfor bacteria (e.g., a treatment that inhibits growth of and/or thatkills bacteria). For example, in some embodiments, the present inventionprovides a composition comprising an anti-infective peptide (i.e., apeptide that inhibits growth or that is capable of killing bacteria(e.g., Gram positive bacteria)) and other type of anti-infectivepharmaceutical or compound (e.g., non peptide anti-infective). In someembodiments, the anti-infective peptide is a cationic peptide. In someembodiments, the anti-infective peptide is a defensin. In someembodiments, the anti-infective peptide is a lantibiotic (e.g., nisin).In some embodiments, the second anti-infective agent is a small moleculeantibiotic. In some embodiments, the second anti-infective agent is anantibiotic that alters bacterial metabolism (e.g., an antibiotic thatinterferes with cellular metabolism and/or RNA synthesis and/or proteinsynthesis). In some embodiments, the second anti-infective agent is anamino glycoside (e.g., gentimycin). In some embodiments, the secondanti-infective agent is mupirocin. Although an understanding of themechanism is not necessary to practice the present invention and thepresent invention is not limited to any particular mechanism of action,it is contemplated that a composition comprising an anti-infectivepeptide is capable of generating pores in bacteria thereby enhancing theentry of compounds (e.g., small molecule antibiotic or metabolismantibiotic) into bacteria. Although an understanding of the mechanism isnot necessary to practice the present invention and the presentinvention is not limited to any particular mechanism of action, it iscontemplated that the compositions of the present invention maystimulate (e.g., when in contact with the skin) a host defense response(e.g., an innate immune response) thereby decreasing the likelihood ofor reducing bacterial infection.

In other preferred embodiments, the present invention provides acomposition comprising nisin and mupirocin. In some embodiments, acomposition comprising nisin and mupirocin is administered to a subjectunder conditions such that bacteria (e.g., pathogenic bacteria) arekilled. In some embodiments, a composition comprising nisin andmupirocin is administered to a subject under conditions such thatbacteria (e.g., pathogenic bacteria) growth is prohibited and/orattenuated. In some embodiments, greater than 90% (e.g., greater than95%, 98%, 99%, all detectable) of bacteria are killed. In someembodiments, there is greater than 2 log (e.g., greater than 3 log, 4log, 5 log, or more) reduction in bacteria. In some embodiments, thereduction is observed in two days or less following initial treatment(e.g., less than 24 hours, less than 20 hours, 18 hours or less). Insome embodiments, the reduction is observed in three days or less, fourdays or less, or five days or less.

The present invention demonstrates that a composition comprising alantibiotic (e.g., nisin) and mupirocin is more efficacious (e.g., insome embodiments, provides an additive effect, and in other embodiments,provides a synergistic effect) at treating skin infections (e.g.,killing bacteria or prohibiting bacterial growth (e.g., of subcutaneousskin infections or deep wound infections)) when compared to either agentalone (See Example 1). The specific efficacy of a composition comprisinga lantibiotic (e.g., nisin) and mupirocin compared to a lantibiotic(e.g., nisin) plus other type of anti-bacterial agent was observedduring the development of the present invention. For example, when nisinwas combined with two other anti-bacterial agents separately(lysostaphin and bacitracin), no additive or synergistic effect wasobserved. Thus, in some embodiments, the present invention provides acomposition comprising mupirocin and a lantibiotic (e.g., nisin).

A composition comprising mupirocin and a lantibiotic (e.g., nisin) ofthe present invention can be administered (e.g., to a subject (e.g., tothe skin or other surface of a subject) or to bacteria (e.g., pathogenicbacteria (e.g., residing on or within the skin of a subject))) as atherapeutic or as a prophylactic to prevent bacterial infection. Thus,in some embodiments, the present invention provides a method of alteringbacterial (e.g., pathogenic bacteria) growth comprising administering acomposition comprising a lantibiotic (e.g., nisin) and mupirocin to thebacteria. In some embodiments, administration of a compositioncomprising a lantibiotic (e.g., nisin) and mupirocin to the bacteriakills the bacteria. In some embodiments, administration of a compositioncomprising a lantibiotic (e.g., nisin) and mupirocin to the bacteriainhibits growth of the bacteria. It is contemplated that a compositioncomprising mupirocin and a lantibiotic (e.g., nisin) can be administered(e.g., to a subject or bacteria) via a number of delivery routes and/ormechanisms.

For example, the compositions of the present invention can beadministered (e.g., to a subject (e.g., to a skin burn or woundsurface)) by multiple methods, including, but not limited to, beingsuspended in a solution (e.g., colloidal solution) and applied to asurface (e.g., a surface comprising bacteria (e.g., pathogenic bacteria)or susceptible to bacterial invasion); being suspended in a solution andsprayed onto a surface using a spray applicator; being mixed with fibringlue and applied (e.g., sprayed) onto a surface (e.g., skin burn orwound); being impregnated onto a wound dressing or bandage and applyingthe bandage to a surface (e.g., an infection or wound); being applied bya controlled-release mechanism; being impregnated on one or both sidesof an acellular biological matrix that can then be placed on a surface(e.g., skin wound or burn) thereby protecting at both the wound andgraft interfaces; being applied as a liposome; or being applied on apolymer.

While an understanding of the mechanism is not necessary to practice thepresent invention and while the present invention is not limited to anyparticular mechanism of action, it is contemplated that, in someembodiments, once administered (e.g., to a subject or to a sitecomprising bacteria (e.g., pathogenic bacteria (e.g., MRSA))),compositions (e.g., a topical pharmaceutical composition) comprisingmupirocin and nisin come into contact with bacteria (e.g., pathogenicbacteria) thereby killing and/or preventing growth of the bacteria.

In some embodiments, compositions and methods of the present inventionfind application in the treatment of surfaces for the attenuation orgrowth inhibition of unwanted bacteria (e.g., pathogens). For example,surfaces that may be used in invasive treatments such as surgery,catheterization and the like may be treated to prevent infection of asubject by bacterial contaminants on the surface. It is contemplatedthat the methods and compositions of the present invention may be usedto treat numerous surfaces, objects, materials and the like (e.g.,medical or first aid equipment, nursery and kitchen equipment andsurfaces) in order to control and/or prevent bacterial contaminationthereon.

In some embodiments, a composition of the present invention may beimpregnated into absorptive materials, such as sutures, bandages, andgauze, or coated onto the surface of solid phase materials, such assurgical staples, zippers and catheters to deliver the compositions to asite for the prevention of microbial infection. Other delivery systemsof this type will be readily apparent to those skilled in the art.

Other uses for a composition comprising mupirocin and nisin of theinvention are also contemplated. These include a variety ofagricultural, horticultural, environmental and food processingapplications. For example, in agriculture and horticulture, variousplant pathogenic bacteria may be targeted in order to minimize plantdisease. One example of a plant pathogen suitable for targeting isErwinia amylovora, the causal agent of fire blight.

The compositions of the invention may be formulated for administrationby any route, such as oral, topical or parenteral. The compositions maybe in the form of tablets, capsules, powders, granules, lozenges, creamsor liquid preparations, such as oral or sterile parenteral solutions orsuspensions.

The topical formulations of the present invention may be presented as,for instance, ointments, creams or lotions, foams, eye ointments and eyeor ear drops, impregnated dressings and aerosols, and may containappropriate conventional additives such as preservatives, solvents toassist drug penetration, and emollients in ointments and creams.

The topical formulations may also include agents that enhancepenetration of the active ingredients through the skin. Exemplary agentsinclude a binary combination of N-(hydroxyethyl)pyrrolidone and acell-envelope disordering compound, a sugar ester in combination with asulfoxide or phosphine oxide, and sucrose monooleate, decyl methylsulfoxide, and alcohol.

Other exemplary materials that increase skin penetration includesurfactants or wetting agents including, but not limited to,polyoxyethylene sorbitan mono-oleoate (Polysorbate 80); sorbitanmono-oleate (Span 80); p-isooctyl polyoxyethylene-phenol polymer (TritonWR-1330); polyoxyethylene sorbitan tri-oleate (Tween 85); dioctyl sodiumsulfosuccinate; and sodium sarcosinate (Sarcosyl NL-97); and otherpharmaceutically acceptable surfactants.

In certain embodiments of the invention, the formulations may furthercomprise one or more alcohols, zinc-containing compounds, emollients,humectants, thickening and/or gelling agents, neutralizing agents, andsurfactants. Water used in the formulations is preferably deionizedwater having a neutral pH. Additional additives in the topicalformulations include, but are not limited to, silicone fluids, dyes,fragrances, pH adjusters, and vitamins.

The topical formulations may also contain compatible conventionalcarriers, such as cream or ointment bases and ethanol or oleyl alcoholfor lotions. Such carriers may be present as from about 1% up to about98% of the formulation. The ointment base can comprises one or more ofpetrolatum, mineral oil, ceresin, lanolin alcohol, panthenol, glycerin,bisabolol, cocoa butter and the like.

In some embodiments of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product.

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, preferablydo not unduly interfere with the biological activities of the componentsof the compositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents (e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like) that do not deleteriouslyinteract with mupirocin and nisin of the formulation.

In some embodiments, the invention provides a pharmaceutical compositioncontaining (a) a composition comprising mupirocin and a lantibiotic(e.g., nisin); and (b) one or more other agents (e.g., an antibiotic).Examples of other types of antibiotics include, but are not limited to,almecillin, amdinocillin, amikacin, amoxicillin, amphomycin,amphotericin B, ampicillin, azacitidine, azaserine, azithromycin,azlocillin, aztreonam, bacampicillin, bacitracin, benzylpenicilloyl-polylysine, bleomycin, candicidin, capreomycin,carbenicillin, cefaclor, cefadroxil, cefamandole, cefazoline, cefdinir,cefepime, cefixime, cefinenoxime, cefinetazole, cefodizime, cefonicid,cefoperazone, ceforanide, cefotaxime, cefotetan, cefotiam, cefoxitin,cefpiramide, cefpodoxime, cefprozil, cefsulodin, ceftazidime,ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cephacetrile,cephalexin, cephaloglycin, cephaloridine, cephalothin, cephapirin,cephradine, chloramphenicol, chlortetracycline, cilastatin, cinnamycin,ciprofloxacin, clarithromycin, clavulanic acid, clindamycin, clioquinol,cloxacillin, colistimethate, colistin, cyclacillin, cycloserine,cyclosporine, cyclo-(Leu-Pro), dactinomycin, dalbavancin, dalfopristin,daptomycin, daunorubicin, demeclocycline, detorubicin, dicloxacillin,dihydrostreptomycin, dirithromycin, doxorubicin, doxycycline,epirubicin, erythromycin, eveminomycin, floxacillin, fosfomycin, fusidicacid, gemifloxacin, gentamycin, gramicidin, griseofulvin, hetacillin,idarubicin, imipenem, iseganan, ivermectin, kanamycin, laspartomycin,linezolid, linocomycin, loracarbef, magainin, meclocycline, meropenem,methacycline, methicillin, mezlocillin, minocycline, mitomycin,moenomycin, moxalactam, moxifloxacin, mycophenolic acid, nafcillin,natamycin, neomycin, netilmicin, niphimycin, nitrofurantoin, novobiocin,oleandomycin, oritavancin, oxacillin, oxytetracycline, paromomycin,penicillamine, penicillin G, penicillin V, phenethicillin, piperacillin,plicamycin, polymyxin B, pristinamycin, quinupristin, rifabutin,rifampin, rifamycin, rolitetracycline, sisomicin, spectrinomycin,streptomycin, streptozocin, sulbactam, sultamicillin, tacrolimus,tazobactam, teicoplanin, telithromycin, tetracycline, ticarcillin,tigecycline, tobramycin, troleandomycin, tunicamycin, tyrthricin,vancomycin, vidarabine, viomycin, virginiamycin, BMS-284,756, L-749,345,ER-35,786, S-4661, L-786,392, MC-02479, Pep5, RP 59500, and TD-6424. Insome embodiments, two or more combined agents (e.g., a compositioncomprising mupirocin and nisin and another antibiotic) may be usedtogether or sequentially. In some embodiments, another antibiotic maycomprise bacteriocins, type A lantibiotics, type B lantibiotics,liposidomycins, mureidomycins, alanoylcholines, quinolines,eveminomycins, glycylcyclines, carbapenems, cephalosporins,streptogramins, oxazolidonones, tetracyclines, cyclothialidines,bioxalomycins, cationic peptides, and/or protegrins. In someembodiments, the composition comprises lysostaphin.

The present invention also includes methods involving co-administrationof compounds comprising mupirocin and a lantibiotic (e.g., nisin) withone or more additional active agents (e.g., an antibiotic, anti-oxidant,etc.). Indeed, it is a further aspect of this invention to providemethods for enhancing prior art therapies and/or pharmaceuticalcompositions by co-administering a composition comprising mupirocin andnisin of this invention. In co-administration procedures, the agents maybe administered concurrently or sequentially. In one embodiment, thecompounds described herein are administered prior to the other activeagent(s). The pharmaceutical formulations and modes of administrationmay be any of those described herein. In addition, the two or moreco-administered agents may each be administered using different modes ordifferent formulations.

The additional agents to be co-administered, such as other antibiotics,can be any of the well-known agents in the art, including, but notlimited to, those that are currently in clinical use.

Treatment of the various diseases and disorders described herein areoften generally limited by the following two major factors: (1) thedevelopment of drug resistance and (2) the toxicity of known therapeuticagents. Some therapeutic agents have deleterious side effects, includingnon-specific toxicity.

The methods described herein address both these problems. Drugresistance, where increasing dosages are required to achieve therapeuticbenefit, is overcome by co-administering the compounds comprisingmupirocin and a lantibiotic (e.g., nisin) described herein with orwithout a known agent. In some embodiments, the compounds describedherein sensitize target cells to known agents (and vice versa) and,accordingly, less of these agents are needed to achieve a therapeuticbenefit.

The sensitizing function of the claimed compositions also addresses theproblems associated with toxic effects of known therapeutics. Ininstances where the known agent is toxic, it is desirable to limit thedosages administered in all cases, and particularly in those cases wheredrug resistance has increased the requisite dosage. Thus, in someembodiments, when the claimed compounds are co-administered with theknown agent, they reduce the dosage required which, in turn, reduces thedeleterious effects. Further, because the claimed compounds arethemselves both effective and non-toxic in moderate doses,co-administration of proportionally more of these compounds than knowntoxic therapeutics will achieve the desired effects while minimizingtoxic effects.

In some embodiments, pharmaceutical preparations comprising compositionscomprising mupirocin and nisin are formulated in dosage unit form forease of administration and uniformity of dosage. Dosage unit form, asused herein, refers to a physically discrete unit of the pharmaceuticalpreparation appropriate for the patient undergoing treatment. Eachdosage should contain a quantity of the compositions comprisingmupirocin and a lantibiotic (e.g., nisin) calculated to produce thedesired antibacterial (e.g., killing or growth attenuation of bacteria)effect in association with the selected pharmaceutical carrier.Procedures for determining the appropriate dosage unit are well known tothose skilled in the art.

Dosage units may be proportionately increased or decreased based on theweight of the patient. Appropriate concentrations for achievingeradication of pathogenic bacteria in a target cell population or tissuemay be determined by dosage concentration curve calculations, as knownin the art.

In some embodiments, the composition comprises from 0.1 to 2000 μg/mL oflantibiotic (e.g., nisin) and 0.1 to 2000 μg/mL mupirocin (e.g., 1-1000μg/mL, 1-500 μg/mL, 5-200 μg/mL etc.). In some embodiments, thecomposition is from 0.01 to 15% (e.g., 0.1-10%, 0.5-5%, 1-3%, 2%) byweight lantibiotic (e.g., nisin) and/or mupirocin. In some embodiments,the amount of lantibiotic (e.g., nisin) and/or mupriocin delivered to asubject is from 0.1 to 1000 mg/kg/day (e.g., 1 to 500 mg/kg/day, 5 to250 mg/kg/day, 10-100 mg/kg/day, etc.). In some embodiments, the ratioof lantibiotic (e.g., nisin) concentration to mupirocin concentration is10:1, 5:1, 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, 1:5, 1:10, etc.

It is contemplated that the compositions and methods of the presentinvention will find use in various settings, including researchsettings. For example, compositions and methods of the present inventionalso find use in studies of antibiotic resistance (e.g., via analysis ofproteins and pharmaceuticals capable of altering antibiotic resistance)and in in vivo studies to observe susceptibility of bacterial cells toantibacterial treatments. Uses of the compositions and methods providedby the present invention encompass human and non-human subjects andsamples from those subjects, and also encompass research applicationsusing these subjects. Thus, it is not intended that the presentinvention be limited to any particular subject and/or applicationsetting.

The compositions of the present invention find use where the nature ofthe infection present or to be avoided is known, as well as where thenature of the infection is unknown. For example, the present inventioncontemplates use of the compositions of the present invention intreatment of or prevention of infections associated with any topicalapplication involving ailments of the skin, including, but not limitedto, skin lesions, wounds, ulcers, bed sores, diaper rash, blisters,acne, psoriasis, and warts.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1 Compositions comprising mupirocin and nisin clear mouse skininfection

Materials: Lysostaphin was made by Biosynexus, Inc. and Nisin (AMBICINN) was obtained from AMBI, Inc. Mupirocin (BACTROBAN Ointment),bacitracin (Sigma), and Bacitracin Ointment (G&W Laboratories Inc) wereall obtained commercially. Polyethylene glycol (PEG) 400 and PEG 3350were purchased from Spectrum Chemicals.

Mouse Skin Infection Model: An overnight culture of S. aureus grown intryptic soy broth (SA8; range from 1 to 6×10⁹ CFUs/mL) was centrifugedat 4000×g for 10 minutes and resuspended in an equal volume of phosphatebuffered saline (PBS). The bacteria were diluted to a percenttransmittance of 40 (Spectronic 20D+) and diluted a subsequent 1:1000 inPBS for a final bacteria concentration of about 3×10⁵. SKH1 (hrhr)hairless mice (Charles River) were sedated with 0.2 mL of ketamine (80mg/kg) and xylazine (32 mg/kg) delivered intraperitoneally. The upperback of the mice were scrubbed with a 70% alcohol wipe and allowed todry. Fine abrasions were made on the backs of the mice between theshoulders using 150 grit sandpaper. Bacteria were swabbed over theabraded area with a sterile, cotton-tipped applicator until the area wassaturated with the solution.

Topical Ointment: 74 g of PEG 400 and 24 g of PEG 3350 were added to a250 mL glass beaker and heated until all of the PEG 3350 was melted. Thesolution was stirred well and allowed to cool to room temperature, whichresulted in a smooth, opaque ointment. Powdered nisin and bacitracinwere added to the ointment on a w/w basis and stirred until homogenouslymixed. For formulations containing mupirocin, 1 g of Bactroban Ointmentwas weighed into a container and powdered nisin added to 2% or 6% (w/w)and mixed well. Lysostaphin was first dissolved to a concentration ofabout 150 mg/mL in DI water before mixing into the ointment to give afinal concentration of 2% (w/w).

Treatment of Infected Skin with Topical Ointment: Treatments aimed toeradicate S. aureus skin infections in the mouse model were started onthe morning after infection (Day 1). About 0.1 g of the topicalointments containing nisin (0, 2, or 6% w/w), mupirocin (0 or 2%),bacitracin (0 or 500U), and lysostaphin (0 or 2% w/w) were swabbed overthe infected area 3 times a day on Days 1 and 2 using a sterile,polyester-tipped applicator. The mice were sacrificed by CO₂asphyxiation on the morning of Day 3 and a 0.5-cm² patch of skin aroundthe infected area was excised. The skin sample was disected and placedinto a test tube containing 1 mL of 5 mg/mL proteinase K, 20 mg/mLesterase, and 20 mg/mL activated charcoal in PBS to neutralize theactivity of the antibacterial agents. Bacteria were dislodged from theskin sample by sonication: 2 minute treatments per sample withalternating 5 seconds at 3 W and 5 seconds at 0 W in a Virsonic 600 withmicrotip (VirTis). After mixing by vortex, 50 μL of each sample wasstreaked onto blood agar plates, incubated at 37° C. overnight, and thecolonies were counted and compared to untreated controls.

For the following data, except where noted in FIG. 2, all antibioticformulations were in a common polyethylene glycol (PEG) ointment (PEG400 plus PEG 3350).

FIG. 1 shows that the efficacy of nisin alone increased with increasingnisin doses and decreases infectious CFUs by 2 logs, compared to a 3 logdrop with mupirocin alone (on a molar bases, 2% mupirocin is twice thedose of 6% nisin). Neither drug alone was able to completely clear theinfection. In contrast, 3 of 10 animals in the 2% nisin/2% mupirocincombination group and 5 of 11 in the 6% nisin/2% mupirocin combinationgroup were cleared of infection and the infectious CFUs in the residualinfections were reduced by 5 logs, 100-fold more than either therapyalone.

FIG. 2 shows the efficacy of nisin, lysostaphin, and bacitracin aloneand in combination on S. aureus infection in abraded mouse skin usingtwo different formulations of bacitracin: PEG ointment and acommercially available formulation from G&W Laboratories Inc (petrolatumbase). None of these antibacterial agents administered independently, oradministered in a composition comprising nisin, were significantly moreefficacious than the independent administration of nisin (about 1.5 logreduction).

As shown in FIG. 3, mupirocin exhibited minimal activity against an S.aureus strain resistant to mupirocin. However, nisin was capable ofreducing infectious CFUs by 3 logs. Mupirocin has previously been shownto have poor activity against P. aeruginosa. However, nisin's spectrumof activity includes gram negative bacteria when formulated in thepresence of chelators, surfactants, or essential oils.

As shown in FIG. 4, nisin plus EDTA had more activity than nisin alonein this formulation and reduced infectious CFUs by 1.5 logs. Mupirocindoes not add any additional activity to the nisin formulation.

Example 2 Compositions comprising mupirosin and nisin eliminateinoculated Staphylococcus aureus from partial thickness wounds

Experimental animals. A young female specific pathogen free (SPF: LooperFarms, North Carolina) pig weighing 25-30 kg was kept in house for twoweeks prior to initiating the experiment. The animal was fed a basaldiet ad libitu and housed individually in animal facilities (meetingAmerican Association for Accreditation of Laboratory Animal standards)with controlled temperature (19-21° C.) and lights (12h/12LD). Theexperimental animal protocols used for this study followed the federalguidelines for the care and use of laboratory animals (See U.S.Department of Health and Human Services, U.S. Department ofAgriculture). Animals were monitored daily for any observable signs ofpain or discomfort. In order to help minimize possible discomfort, ananalgesic buprenorphine 0.03 mg/kg (Buprenex injectable; ReckittBenckiser Hull, England) was given to each animal on the first day, andevery third day thereafter, while under anesthesia; a fentanyltransdermal system: 25 μg/hr (Duragesic; Alza Corp. Mountain View,Calif.) were used during the entire experiment.

Animal Preparation, Wounding and Treatment. Each animal was anesthetizedwith Telazol HCl (1.4 mg/kg), Xylazine (2 mg/kg), Atropine (0.05 mg/kg)I.M. and inhalation of an isofluorane and oxygen combination. Hair onthe back of the pig was clipped with standard animal clippers. Skin onboth sides of the animal was prepared by washing with a non-antibioticsoap (NEUTROGENA) and sterile water. The animal was blotted dry withsterile gauze. Forty-eight partial thickness wounds measuring (10 mm×7mm×0.3 mm deep) were made in the paravertebral and thoracic area of eachanimal with a specialized electrokeratome fitted with a 7 mm blade. Thewounds were separated from one another by at least 7 cm of unwoundedskin. Each wound was then inoculated with a known amount ofStaphylococcus aureus (10⁶ suspension). The suspension was lightlyscrubbed into the test site for ten seconds using a sterile TEFLONspatula. After inoculation wounds were covered with TEGADERMpolyurethane film dressing (3M, Inc.) for 24 hours before the initiationof the treatment in order to give the bacteria time to colonize thewounds and develop a biofilm.

Wound Inoculation. A fresh culture of pathogenic isolate obtaineddirectly from American Type Culture Collection (ATCC), Rockville, Md.,was used (Staphylococcus aureus ATCC #6538). The freeze-dried bacteriaculture was recovered per ATCC standard recovering protocol. Allinoculum suspensions were made by scraping the overnight growth from aculture plate into 4.5 ml of sterile water and making up the suspensionuntil the turbidity of the suspension was equivalent to that of aMcFarland #8 Turbidity Standard. This resulted in a suspensionconcentration of approximately 10⁸ colony forming units/ml (CFU/ml). The10⁸ suspension was serially diluted to make an inoculum suspension witha concentration of 10⁶ CFU/ml in 35 mls of Tryptic Soy Broth (TSB). Asmall amount of the inoculum suspension was plated onto culture media toquantitate the exact concentration of viable organisms prior to theexperiment. The inoculum suspension was used directly to inoculate eachsite. A 25 μl aliquot of the suspension was deposited into a sterileglass cylinder (22 mm diameter) in the center of each wound site. Thesuspension was lightly scrubbed into the test site for ten seconds usinga sterile TEFLON spatula. After inoculation the wounds were covered witha polyurethane dressing for 48 hours to allow the bacteria to develop abiofilm on the wounds. Dressings were then removed to culture thebaseline wounds and to treat the rest according to the experimentaldesign described in FIG. 5. Wounds were treated twice per day.

Recovery Methods. Three wounds were cultured 48 hours post inoculationto quantitate the biofilm baseline, and two wounds from each treatmentgroup on Days 2, 5 and 7 post treatment. At each sampling time, siteswere cultured quantitatively. Each site was cultured only once. Thewounded area was encompassed by a sterile glass cylinder (22 mm outsidediameter) held in place by two handles. One mL of scrub solution waspipetted into the glass cylinder and the site was scrubbed with asterile TEFLON spatula for 30 seconds.

Serial dilutions were made and scrub solutions were quantitated usingthe Spiral Plater System that deposits a small defined amount (50 μl) ofsuspension over the surface of a rotating agar plate. The selectivemedia for Staphylococcus aureus was Mannitol Salt Agar. All samples wereincubated aerobically for 24 hours at 37° C. After the incubation period(24 hrs), colonies on the plates were counted and the colony formingunits per mL (CFU/ml) calculated. The presumptive identification testfor the pathogen is the ability of S. aureus to coagulate rabbit plasma.

Three wounds per treatment group were cultured 2, 5 and 7 days posttreatment. They were serially diluted, plated on Mannitol-salt agar, andincubated for 24 hours. After the incubation period colonies werecounted and the Log colony forming units/mL determined. The geometricmean of the Log (CFU/mL) and standard deviation were calculated for eachtime and treatment. The initial inoculum size used for this experimentwas 6.13 log cfu/ml. The baseline counts after 24 hours of inoculationin the wound environment were 7.48±1.0 log cfu/ml. The combined raw datafor the total experiment is shown in FIG. 14. Active X is a compositionhaving nisin (6%). Active Y is a composition having nisin (6%) andmupirocin (2%).

The data comparing treatments day by day was as follows:

Two days after initial treatment of inoculated wounds, a compositioncomprising mupirocin and nisin was observed to completely eliminateStaphylococcus aureus from the wounds (See FIG. 6). The next mosteffective treatments were nisin, and mupirocin, which yielded 2.29±2.0and 4.96±0.8 Log CFU/ml, respectively. The Vehicle-treated wounds andthe wounds left air-exposed yielded similar numbers on this day (6.5 Logcfu/ml and 6.9 Log cfu/ml, respectively) (See FIG. 6).

On Day 5 after treatment, the same trend was observed as the priorassessment point. Bacteria cultured from wounds treated with acomposition comprising mupirocin and nisin yielded no Staphylococcusaureus (See FIG. 7), with nisin being the next most effective treatmentyielding a less than 1 Log cfu/ml. Mupirocin had a similar outcome.Vehicle-treated wounds yielded about 3 log cfu/ml of bacteria less thanthe negative control group, which had about 4.5 log cfu/ml bacteria (SeeFIG. 7).

Day 7 post treatment, the final day of assessment, the trend observed inthe previous two time points remained. Wounds treated with a compositioncomprising mupirocin and nisin yielded no Staphylococcus aureus (SeeFIG. 8), while nisin yielded 1.05±1.8 Log cfu/ml. Mupirocin yielded 1.63Log cfu/ml, which was close to the Vehicle results (1.77±1.6 LogCFU/ml). The negative control resulted in a little less than 4 logcfu/ml (3.7) (See FIG. 8). A summary of the data from all threetreatment timepoints is depicted in FIG. 9.

Example 3 Compositions comprising gentimycin and nisin eliminatesStaphylococcus aureus

Mice provided a superficial skin abrasion and inoculated with S. aureusas described in Example 2 were treated with a composition comprising acombination of 6% nisin and 0.1% gentamicin. This combination eliminatedsubstantially all detectable S. aureus.

Example 4 A composition comprising nisin and mupirocin is superior to acomposition comprising only mupirocin in treating methicillin sensitiveand methicillin resistant S. aureus

Mice were provided a superficial skin abrasion as described in Example 2and were infected with either methicillin sensitive S. aureus (MSSA) ormethicillin resistant S. aureus (MRSA) and then treated with acomposition comprising: vehicle (PEG cream) alone, mupirocin (2%), ormupirocin (2%) and nisin (6%) (Active Y). FIG. 10 shows the mean log(CFU/ml) of bacteria recovered after either a first or second treatment(MSSA) or after one and three days (MRSA). The combination of nisin andmupirocin was superior in treating (e.g., killing and/or inhibitinggrowth of) both MSSA and MRSA compared to controls and either treatmentalone.

Example 5 Nisin does not synergize with mupirocin, neomycin, orgentamicin in vitro

In order to determine if nisin could function in a synergistic way withother antimicrobials to treat (e.g., kill and/or inhibit growth of)bacteria in vitro, the minimum inhibitory concentration (MIC) of nisinalone or nisin in combination with either mupirocin, neomycin orgentamicin were tested. The MIC was determined by calculating the logreduction of viable S. aureus following incubation with the amounts ofnisin and other antimicrobials as indicated in FIG. 11.

As documented in FIG. 11, none of the antimicrobials tested (i.e.,mupirocin, neomycin, and gentamicin) displayed the ability to synergizein vitro with nisin.

Example 6 Nisin does not synergize with neomycin or gallidermin intreating skin infection

In order to determine if nisin could function in a synergistic way withother antimicrobials to treat (e.g., kill and/or inhibit growth of)bacteria in a superficial wound model (See Example 2), nisin was used incombination with either neomycin or gallidermin and tested. Nisin didnot synergize with neomycin nor with gallidermin in the skin infectionmodel.

Example 7 Nisin does synergize with gentamicin and gallidermin doessynergize with mupirocin in treating skin infection

In order to determine if nisin or gallidermin could function in asynergistic way with other antimicrobials to treat (e.g., kill and/orinhibit growth of) bacteria in a superficial wound model (See Example2), nisin or gallidermin were used in combination with either gentamicinand mupirocin, respectively, and tested. Nisin did synergize withgentamicin and gallidermin did synergize with mupirocin to reducebacterial counts in the skin infection model.

Example 8 Mupirocin does not synergize with bacitracin in treating skininfection

In order to determine if mupirocin could function in a synergistic waywith bacitracin to treat (e.g., kill and/or inhibit growth of) bacteriain a superficial wound model (See Example 2), mupirocin was used incombination with bacitracin and tested for the ability to treatbacteria. As shown in FIG. 12, mupirocin did not synergize withbacitracin, nor was there an additive benefit when the two were usedtogether in the reduction of S. aureus in the infected wound model.

Example 9 A composition comprising nisin and mupirocin provide asynergistic ability to treat S. aureus in a suture infection model

It was determined whether nisin and mupirocin, either alone or incombination with each other, would be able to treat S. aureus in a deeptissue infection model. A suture skin infection model was generated inwhich a deep cut (e.g., one needing sutures to close) was made in amouse and S. aureus introduced into the incision. As shown in FIG. 13,nisin and mupirocin alone show very little efficacy in treating the S.aureus. However, the combination of nisin plus mupirocin nearlyeradicated the infection. Thus, a combination of nisin and mupirocin canbe used to treat a deep tissue infection as well as a subcutaneousinfection.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in therelevant fields are intended to be within the scope of the followingclaims.

1. A pharmaceutical formulation comprising nisin and mupirocin.
 2. Thepharmaceutical formulation of claim 1, comprising 6% nisin and 2%mupirocin.
 3. The pharmaceutical formulation of claim 2, wherein saidformulation provides a more than additive killing and/or growthinhibition of bacterial cells administered the formulation compared toindividual administration of either nisin or mupirocin.
 4. Thepharmaceutical formulation of claim 3, wherein said killing and/orinhibiting bacterial cells occurs within an existing bacterialinfection.
 5. The pharmaceutical formulation of claim 3, wherein saidbacterial cells comprise bacteria of the genus Staphylococcus.
 6. Thepharmaceutical formulation of claim 5, wherein said bacterial cellscomprise Staphylococcus aureus.
 7. The pharmaceutical formulation ofclaim 6, wherein said Staphylococcus aureus are pathogenic
 8. Thepharmaceutical formulation of claim 6, wherein said Staphylococcusaureus comprise drug resistant Staphylococcus aureus.
 9. Thepharmaceutical formulation of claim 6, wherein said Staphylococcusaureus are methicillin resistant.
 10. The pharmaceutical formulation ofclaim 1, further comprising a pharmaceutically acceptable carrier. 11.The pharmaceutical formulation of claim 10, wherein said carriercomprises polyethylene glycol.
 12. The pharmaceutical formulation ofclaim 10, formulated for administration to a wound.
 13. Thepharmaceutical formulation of claim 10, wherein said pharmaceuticalformulation is a cream.
 14. The pharmaceutical formulation of claim 10,wherein said pharmaceutical formulation is a spray.
 15. Thepharmaceutical formulation of claim 10, wherein said pharmaceuticalformulation is prepared for controlled time release.